Dosage compensation (DC) is an essential process required to balance levels of gene expression between the two X chromosomes of females and the single X of males. In the nematode C. elegans, as in flies and mammals, DC modulates gene expression across an entire chromosome and thereby serves as an exemplary system to understand long-range mechanisms of gene regulation. To enact DC in C. elegans, a dosage compensation complex (DCC) is recruited to the X chromosome by recruitment elements, caled rex sites. The DCC also binds regions of X that cannot independently recruit the DCC, called dox sites, suggesting a model in which the DCC is recruited to rex sites and spreads to dox sites. In addition, not all genes on X are dosage compensated and binding of the DCC to a particular gene is not predictive of whether that gene will be dosage compensated. Thus, the DCC acts at a distance to control gene expression. How recruitment sites function to recruit the DCC, facilitate spreading to dox sites, and control gene expression is unknown. Progress here requires the identification of dosage compensated genes with high confidence and resolution along X and the manipulation of rex sites in their endogenous context. Site-directed mutagenesis in vivo using zinc-finger and TALE nucleases and Mos1- mediated single copy insertion will be employed to delete and insert rex sites in the context of the X chromosome to explore the effects of individual and groups of rex sites on DCC binding. Whole-transcriptome sequencing of wild-type and rex-mutant worms will be used to determine the effect of individual rex sites on gene expression and DC. Finally, gene expression of a reporter will be assessed when moved throughout the X chromosome in different epigenetic contexts and at different distances from endogenous and engineered rex sites to identify the parameters that confer DC status. Together, these experiments will uncover the mechanisms by which regulatory elements on X enact appropriate patterns of gene expression chromosome- wide and will provide insights into how genomic organization contributes affects gene expression. PUBLIC HEALTH RELEVANCE: In many cancers, genes that control cell proliferation are silenced by changes in chromosome structure that affect large chromosomal territories, allowing cancer cells to evade appropriate proliferative control. Dosage compensation, which reduces gene expression across an entire chromosome, is an ideal model for dissecting the mechanisms of long-range gene silencing like those that occur in cancer cells.